Nucleic acid and allergenic polypeptides encoded thereby in cashew nuts

ABSTRACT

The invention describes an isolated nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO:1 or a degenerate variant of SEQ ID NO: 1. The nucleic acid sequence encodes an Ig-E binding immunogenic polypeptide the amino acid sequence of which comprises at least one sequence selected from SEQ ID NOS:4-25. The invention additionally provides an in vitro diagnostic test for detecting anti-cashew IgE in a patient. The test comprises reacting the patient&#39;s serum with a purified polypeptide the amino acid sequence of which comprises at least one sequence selected from SEQ ID NOS:3-25; separating the polypeptide from unreacted patient serum; reacting the polypeptide with a labeled human IgE-reactive agent after separating from unreacted patient serum; separating the polypeptide from unreacted labeled human IgE-reactive agent; and detecting labeled human IgE-reactive agent bound to the polypeptide after separating from unreacted agent to thereby indicate presence in the patient&#39;s serum of anti-cashew IgE.

RELATED APPLICATION

This application claims priority from co-pending U.S. provisionalapplication Ser. No. 60/423,556, which was filed on Nov. 4, 2002, andwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of cell biologyand, more particularly, to a nucleic acid sequence and the polypeptidesencoded thereby which contain an allergen found in cashew nuts.

BACKGROUND OF THE INVENTION

Allergic reactions to cashew nuts (Anacardium occidentale), thoughrelatively infrequent, can be varied and even life threatening reactionsinclude contact or systemic dermatitis (to cardol and anacardic acidfound in the cashew nut shell oil) (Marks et al. 1984), atopicdermatitis, and IgE-mediated systemic allergic reactions. (Burks et al.1998; Tariq et al. 1996; Garcia et al. 2000) Pistachios and mangos areother edible members of the Anacardiaceae family and are allergenic withpistachio showing extensive in vitro and possible clinicalcross-reactivity with cashews. (Fernandez, Fiandor, Martinez-Garate, andMartinez 1995) (Parra et al. 1993) (Quercia, Rafanelli, Marsigli,Foschi, and Stefanini 1999). Cashew nuts are widely used in snack foodsand as an ingredient in a variety of processed foods such as “butters”,bakery and confectionery products.

We have recently demonstrated that the major IgE-reactive proteins inextracts of cashew nut are legumin-like proteins and 2S albumins asassessed by N-terminal and enzymatic fragment sequencing of nativeproteins. (Teuber, Sathe, Peterson, and Roux 2002) We have alsoconstructed a cashew nut cDNA library and, upon initial screening,cloned and sequenced an allergen in the 7S superfamily, which includesvicilin-like and sucrose binding proteins. (Wang et al. 2002) Here, wepresent the sequence and characteristics of a second cDNA encodingprotein, designated Ana o 2, which is a member of the legumin (11Sglobulin) family of seed storage proteins. We also describe its reactionwith sera from cashew-allergic patients and map its linear epitopes.

REFERENCES CITED

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SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention describes a cloned,expressed and characterized a major cashew allergen. A cashew cDNAlibrary was screened with human IgE and rabbit IgG anti-cashew extractantisera, and a reactive non-vicilin clone, sequenced and expressed as afusion protein in E. coli. Immunoblotting was used to screen forreactivity with patient's sera and inhibition of immunoblotting was usedto identify the corresponding native peptides in cashew nut extract. Theidentified allergen was subjected to linear epitope mapping using theSPOTs solid phase synthetic peptide technology.

Sequence analysis showed the selected clone, designated Ana o 2, to be amember of the legumin family (an 11S globulin) of seed storage proteins.By immunoblotting, 13 of 21 (62%) of sera from cashew allergic patientswere reactive. Immunoblot inhibition data showed the native Ana o 2constitutes a major band at ˜33 kD and a minor band at ˜55 kD. Probingof overlapping synthetic peptides with pooled human cashew-allergic seraidentified 22 reactive peptides, 7 of which gave strong signals. SeveralAna o 2 epitopes were shown to overlap those of the peanut allergeniclegumin, Ara h 3, in position but with little sequence similarity.Greater positional overlap and identity was observed between Ana o 2 andsoybean glycinin epitopes. Conclusion: We conclude that thislegumin-like protein is a major allergen in cashew nuts.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings inwhich:

FIG. 1 shows the nucleotide and derived amino acid sequence analysis ofAna o 2 cDNA clone Ana o 2; this figure includes subfigures A1 and 2, B,C, and D, all of which may be collectively referred to as FIG. 1, andwherein

FIGS. 1 A1 and 1A2, respectively, show the nucleotide sequence encodingfor the polypeptide of SEQ ID NO:1, referred to as Ana-o-2, including astop codon shown underlined and in bold; and the polypeptide of SEQ IDNO:2, which is a tail for the polypeptide of SEQ ID NO:1;

FIG. 1 B shows the derived amino acid sequence of the Ana o 2 codingregion (SEQ ID NO:1) shown with the presumed signal sequence underlined,sites of sequence insertions shown in bold text, deletions shown indouble underline in the nine most similar sequences in GenBank (seeTable I) as compared to Ana o 2 indicated; sequences similar topreviously reported cashew legumin group proteins derived by amino acidsequencing, (Teuber 2002) are shown in bold italics;

FIG. 1 C shows details on the sites of insertion (red) and Deletions(blue) as described herein; a vertical line between residues indicatesthe sites of insertions in similar peptides with the insertion size(number of amino acids) and plant species (two or three letter code)shown above the insertion site; deletions are underlined or overlined,and only deletions and insertions of three or more residues areincluded; abbreviations used are as follows: Rc, Ricinus communis; Cav,Corylus avellana; Qr, Quercus robur; Ahy, Amaranthus hypochondriacus;Si, Sesamum indicum; Ms, Magnolia salicifolia; Vs, Vicia sativa; Cp,Cucurbita pepo; Car, Coffea arabica; Ah3, Arachis hypogaea (Ara h 3);and Ah4, Arachis hypogaea (Ara h 4); and

FIG. 1 D shows a comparison of cDNA-derived Ana o 2 sequences to similarsequences of previously reported cashew legumin group proteins derivedby amino acid sequencing. (Teuber et al. 2002) Identical residues are inbold. Accession number: AF453947;

FIG. 2 shows the identification of native Ana o 2 peptide in cashewextract by means of Western blotting, wherein nitrocellulose blots oftotal cashew extract (A) and rAna o 2 (B) were probed with serum fromcashew-allergic patient (#9) and tagged with 125I-labeled anti humanIgE; the first (left) lane in each blot was incubated withoutinhibition; the second through fourth lanes were incubated with rAna o2, rAna o 1 and/or MBP as indicated; the right lanes were incubated withcontrol human sera; arrows indicate locations of native Ana o 1, top,Ana o 2 (acidic subunit), bottom, and an, as yet, unidentified band,middle; the lower and upper bands in (B) representing the uncleaved(precursor) form of rAna o 2 in its monomer and presumptive dimerconfigurations, respectively; and

FIG. 3 shows a comparison of the IgE-binding epitopes of various 11Sglobulins to those of the cashew legumin, Ana o 2; bracketed andnumbered regions indicate locations of the reactive peptides in Ana o 2(also listed in TABLE III); shaded segments indicate regions expressingone or more linear epitopes on the different allergens; identical aminoacids are indicated with a “.” while similar residues are depicted witha “|”; the symbol “/” shows where a portion of a sequence was deletedand a “-” represents where a space was added in the sequence to maximizealignment; the presumed cleavage site between the acidic and basicsubunits (between amino acids 271 and 272) is shown in text above thesequence; boxes denote regions where numerous insertions and deletionsare found when comparing multiple legumin sequences (see FIG. 1C).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including any definitions,will control. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting.Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Moreover, these illustrated embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Otherfeatures and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

Abbreviations Used

Amino acids: the standard one-letter abbreviations are used.

DTT: dithiothreitol.

HRP; horseradish peroxidase.

Nucleotides: the standard one-letter abbreviations are used.

PCR: polymerase chain reaction.

PBS: phosphate buffered saline.

DS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis.

TBS: tris-buffered saline.

Methods

Human Sera

Blood samples were drawn after informed consent from patients withlife-threatening systemic reactions to cashew nut and the sera frozen at−70° C. until use. The study was approved by the human subjects reviewcommittee of the University of California at Davis. The presence ofcashew-reactive IgE was confirmed by Pharmacia ImmunoCAP assay(Pharmacia, Uppsala, Sweden) or by Western immunoblotting. Control serawere obtained from patients with a history of anaphylaxis to walnut,pistachio or hazelnut who reported tolerance of cashews.

Cashew Protein Extract

An albumin/globulin extract was prepared as previously described.Protein concentrations were measured by use of the Bradford proteinassay (BioRad Laboratories, Inc., Hercules, Calif.) using bovine serumalbumin as the standard protein.

Production of Rabbit Polyclonal Antiserum

A rabbit was immunized with 5 mg of cashew extract in Freund's completeadjuvant and boosted four weeks later with 5 mg of cashew extract inincomplete Freund's adjuvant. The rabbit was subsequently bled and theserum stored at −20° C. Guidelines for animal care and welfare as in the“Guide for the Care and Use of Laboratory Animals” prepared by theInstitute of Laboratory Animal resources, National Research Council,National Academy Press, revised 1996, were followed.

Construction and IgE Immunoscreening of Cashew CDNA Library

Library construction and immunoscreening have previously been describedin detail. (Wang 2002) Briefly, cashew nuts in late maturation werechopped, frozen, and ground. Total RNA was extracted in TRIzol (GIBCOBRL Life Technologies Inc., Rockville, Md.) and mRNA was isolated usinga PolyATtract kit (Promega, Madison, Wisc.). The cDNA library wasconstructed using the Uni-ZAP XR Gigapack Cloning Kit (Stratagene Inc.,Cedar Creek, Tex.) and cloned into the lambda Uni-ZAP XR expressionvector. The library was amplified in E. coli strain XL1-Blue andscreened with rabbit (IgG) and human (IgE) antisera. The immunopositiveclones were picked, plaque-purified, and stored in SM buffersupplemented with 2% chloroform at 4° C.

Sequencing and Corresponding Analysis of Selected Genes

Inserts from the selected phage clones were amplified with M13 forwardand reverse primers by PCR. Both strands of the PCR products were thensequenced on an ABI 3100 Genetic Analyzer (Foster City, Calif.) usingcapillary electrophoresis and Version 2 Big Dye Terminators as describedby the manufacturer. Similarity searches and alignments of deduced aminoacid sequences were performed on Genetics Computer Group (GCG) software(Accelrys, Inc. San Diego, Calif.) using the BLAST 2.0 program(www.ncbi.nim.nih.gov/BLAST/).

Cloning, expression and purification of cDNA-encoded proteins. Aspreviously described in detail (Wang et al. 2002), cDNA coding sequenceswere modified by the addition of a Sal I site at 5′ end and a Pst I siteat 3′end by PCR using PfuTurbo DNA polymerase (Stratagene Inc., CedarCreek, Tex.) followed by digestion with Sal I and Pst I and ligation tomaltose binding protein (MBP) fusion expression vector pMAL-c2 (NewEngland BioLabs Inc., Beverly, Mass.), into which a thrombin cleavagesite had been engineered.

For expression, competent E. coli BL21 (DE3) cells (Novagen Inc.,Madison, Wis.) were transformed. Positive clones were identified by PCRscreening using cDNA sequence-specific primers. Single colonies weregrown and induced with isopropyl-D-thiogalactopyranoside (IPTG). Thecells were harvested, lysed with mild sonication, and centrifuged at12,000 g. The lysate supernatant was passed over an amylose affinitycolumn and the fusion protein eluted with 10 mM maltose. The cDNA insertwas produced by PCR amplification of the portion of the sequenceextending from the presumed first codon following the leader peptidethrough the last codon prior the stop codon using the primersTCTAGAGTCGACCGCCAGGAATGGCAAC (forward) andGCTTGCCTGCAGTTAAGCATCATCCCTCATG (reverse). Fusion proteins were cleavedwith thrombin (Sigma, St. Louis, Mo.) and the liberated recombinantprotein purified on a Superdex 200 column (Amersham Pharmacia,Piscataway, N.J.) by high performance liquid chromatography (HPLC).Cleaved and un-cleaved recombinant proteins were concentrated and eitherstored (briefly) at 4° C. until use or frozen at −7° C.

Gel Electrophoresis (SDS-PAGE), Immunoblotting, and Inhibition

r Ana 0 2 (at 10 g/4 mm width) or total cashew extract (100 g/4 mmwidth) samples were boiled in reducing sample buffer and subjected toelectrophoresis, immunoblotting and inhibition as previously described(Wang et al. 2002). For inhibition experiments, 10 g of rAna o 2, rAna o1, and/or MBP were pre-incubated with human antiserum (from patient #9at 1:20 dilution) for 1 hr at 37° C. and then incubated withnitrocellulose strips containing blotted cashew extract overnight at 4°C. The strips were then incubated with the appropriate labeled secondantibody and exposed to x-ray film.

Solid-Phase Peptide (SPOTs) Synthesis and Binding to IgE

Based on the derived amino acid sequence of the 457 amino acid Ana o 2protein (including the presumptive leader sequence, amino acids 1-14),57 overlapping 15-amino acid peptides, each offset by eight amino acids,were synthesized. An additional peptide, corresponding to presumptiveN-terminal amino acid, 15-29, was also produced. Peptides weresynthesized on derivatized cellulose sheets using 9-fluorenlymethoxycarbonyl-derived (Fmoc) amino acids (Genosys Biotechnologies, Inc., TheWoodlands, Tex.) and probed as previously described (Wang et al. 2002).Briefly, the peptide-containing membranes were washed in TBS andincubated o/n at RT in blocking solution, washed in TBS-T and incubatedo/n at 4° C. with pooled patients' sera diluted 1:5 (v:v). Thisincubation was followed by three 5-min washes in TBS-T and an o/n (4°C.) incubation with 125I-anti-human IgE (Hycor Biomedical Inc.) diluted1:10 in a mixture of PBS, 5% nonfat dry milk, and 0.05% Tween-20. Afteradditional washes, IgE-peptide reactivity was identified after a 1-weekexposure at −70° C. to x-ray film or a 17 hr incubation with a MolecularDynamics phosphoimaging screen (Kodak). Phosphoimaging data wasquantified using a Storm 860 scanner (Molecular Dynamics) and itsaccompanying software.

Results

cDNA Library Screening and Gene Characterization

The cDNA library was initially screened for reactivity with both rabbitIgG and (cashew-allergic) human IgE. As previously reported, a total of50 clones displaying dual reactivity were selected for further analysis(Wang et al. 2002). The first four clones selected for sequencing allproved to encode one of two vicilin variants and were designated Ana o1.1 and Ana o 1.2. A further analysis of the remaining 46 clones by PCRusing Ana o 1 specific primers showed that 45 of the 46 were Ana o1-positive. The remaining clone, upon sequencing and comparison toGenBank, proved to be homologous with the legumin family of seed storageproteins (FIG. 1A) and was designated, Ana o 2. Table I lists the nineproteins having the highest degrees of identity (45% to 58%) andsimilarity (63% to 74%) to Ana o 2 and include proteins described aslegumin-like, legumin precursor, legumin A precursor, 11S globulin, and11S globulin b-subunit precursor, representing a diversity of tree andplant species (Table I). Also listed are four 11S family proteins withsomewhat lower identity (42%-47%) and similarity (58%-66%) but whichhave previously been identified as legumin allergens. Two of theallergens are from the peanut, Ara h 3 and Ara h 4, and two fromsoybean, the glycinin G1 and G2 subunits.

Protein Sequence Characterization.

Analysis of the nucleotide (FIG. 1A) and deduced amino acid sequences(FIG. 1B) reveal a 457 amino acid open reading frame, a possible startsite at position 93, and possible leader peptide from 1 to 15.Comparison of the nine most similar sequences and the four abovementioned allergens (listed in Table I) with Ana o 2 amino acid sequenceshows clustered sites of insertions (red segments) and deletions (bluesegments). FIG. 1C shows the sites of insertions (blue over andunderlined) and deletions (red vertical lines) of four or more aminoacids in the eleven compared sequences with respect to the Ana o 1sequence. Multiple insertions are seen between amino acid 14 and 23, 104and 121, and 180 and 198 with additional scattered insertions between247 and 271. In contrast to the 24 insertion, only four deletions arepresent, all of which are clustered in the 245 to 270 region.

We have previously reported N-terminal and tryptic peptide sequences forseveral native IgE reactive cashew proteins (Teuber et al. 2002). Fourof these were found to have similarity to the cDNA sequence of Ana o 2(FIG. 1D and green sequence segments in FIG. 1B) though none wasidentical suggesting that Ana o 2 is a member of a larger gene family orthat there is extensive polymorphism at the relevant loci.

Reactivity of the recombinant protein with human IgE and rabbit IgG.

For immunological characterization, we cloned and expressed Ana o 2beginning at nucleotide 47 (after the presumptive leader peptide)through 1375. The DNA segment was ligated into an expression vectordesigned to allow for purification of the recombinant molecule via amannose-binding protein (MBP) fusion domain in conjunction with anamylose affinity column and a thrombin-specific cleavage site. Theresulting ˜93 kD Ana o 2 fusion protein was affinity purified aspreviously described and digested with thrombin to yield a ˜52 kDpeptide as well as the 43 kD MBP (data not shown). Both cleaved anduncleaved peptides were reactive with specific human IgE and rabbit IgG(data not shown).

Recognition of Ana o 2 as an Allergen

The prevalence of reactivity to rAna o 2 among cashew allergic patientsby Western immunoblofting is shown in Table 2. IgE from 13 of 21 (62%)sera from patients with a history of life-threatening reactions tocashews bound the recombinant. In 6 cases, the intensity of the signalwas strong, but was weak in 7 cases. In contrast, 1 of 10 sera frompatients tolerant of cashew but clinically with life-threateningreactions to other tree nuts reacted with the rAna o 2.

Identification of Native Ana o 2 by SDS-PAGE Immunoblotting.

Having established that Ana o 2 is a major cashew allergen, we nextsought to identify the band or bands in a typical total cashewimmunoblot that correspond to the cloned polypeptide storage proteinprecursor. FIG. 2A shows nitrocellulose strips in which the totalsoluble cashew extract has been blotted and probed with serum from anallergic patient #9. The first strip (left lane) shows this serumreacting with three major bands at ˜55, ˜36 and ˜33 kD. The secondthrough fourth lanes were incubated with the same human serapreincubated with rAna o 2, rAna o 1+rAna o 2, and the fusion protein,MBP, respectively. Note that Ana o 2 inhibits the strong band at ˜33 kDand weakens the band at ˜55 kD. The addition of Ana o 1 inhibits theresidual signal at ˜55 kD. MBP has no inhibitory effect as expected. Theremaining band at ˜36 kD is as yet unidentified. The last lane (right)shows no reactivity with a patient having allergies to agents other thancashew.

FIG. 2B shows reactivity of the human sera with rAna o 2 (˜53 kD) andits inhibition with purified rAna o 2 but not rAna o 1 nor MBP. The bandat ˜120 kD is presumed to be a dimer of rAna o 2.

Identification and Recognition of IgE-reactive Linear Epitopes on Ana o2 and Comparison to Peanut and Soybean Legumin Epitopes.

The entire amino acid length of Ana o 2 was studied by probing 58overlapping solid phase synthetic peptides with sera from 12 patientsrandomly assigned to three pools. Collectively, the three pools reactedweakly (phosphoimaging score from 2.0-3.9×10-3) with 12, moderately(4.0-6.9×10-3) with three, and strongly (7.0×10-3) with seven linearIgE-binding epitopes. The 22 reactive epitopes were distributedthroughout the length of the protein (Table III) with 68% ( 15/22) ofall epitopes residing on the presumed acidic chain of the protein,including 86% ( 6/7) of those epitopes that were found to react stronglywith pooled patient IgE. Only two of the identified epitopes were boundby patients' sera from all three pools. Epitope #6 was bound strongly bypools 1 and 3, and moderately by pool 2. Epitope #3 was bound stronglyby pool 1, but moderately by pools 2 and 3.

To compare the linear epitopes of cashew legumin (Ana o 2) to those ofpeanut (Ara h 3) (Rabjohn et al. 1999) and the soybean legumins (G2 andG1 glycinin) (Helm et al. 2000b) (Beardslee et al. 2000), we aligned thesequences using the BLAST 2.0 program and highlighted the correspondinglinear epitopes (FIG. 3). Of the regions included in the BLASTalignment, only two of the 22 Ana o 2 linear epitope-bearing peptides(#13 and 15) showed significant (>7 amino acid) positional overlap withany of the four previously identified peanut Ara h 3 epitope-bearingpeptides (FIG. 3) (Rabjohn et al. 1999). Even among these twooverlapping epitope regions, there was little identity or similaritybetween the Ana o 2 and Ara h 3 (Rabjohn et al. 1999). A much greaterdegree of correspondence was observed when the sequences of Ana o 2 andG2 glycinin were aligned; 9 of the 11 (89%) previously identified G2glycinin epitopes showed significant positional overlap with the Ana o 2epitopes. All 9 showed a significant degree of similarity (60%) and twowere quite similar in sequence to Ana o 2 epitopes #1 (77%) and theadjacent pair, #10 and #11 (80%) (FIG. 3). Two IgE-binding linearepitopes have been identified on G1 glycinin (Beardslee et al. 2000),both on the acidic chain. Sequence alignment revealed that only thesecond glycinin epitope displayed significant positional overlap and nosignificant identity with a cashew legumin epitope (#13). Interestingly,this epitope (#13) is the only one identified that displays positionaloverlap with an epitope in each of the other three compared 11Sglobulins (FIG. 3) suggesting the possibility of a shared structuralmotif favoring immunogenicity.

Comparison of rAna o 2 to Native Peptides

We have recently reported the N-terminal and tryptic peptide sequencesof four legumin proteins bands (Teuber et al. 2002). In comparing thesesequences to those of rAna o 2, we note that none are identical insequence (Table IV). One tryptic peptide matched at 8 of 9 positions.Two other nearly identical tryptic peptides matched rAna o 2 at only 13of 19 position and 12 of 18 positions. An N-terminal peptide matched at7 or 8 of 10 positions beginning at residue 272 of rAna o2. Together,these data suggest that these represent a complex family ofdifferentially processed proteins.

Discussion

Approximately 0.5% of the US population is believed to be allergic totree nuts (Sicherer et al. 1999) and the data from a voluntary registryof peanut and tree nut allergic US patients shows 20% of those reportingallergy to tree nuts list sensitivity to cashews, the highest percentagefor any tree nut. (Sicherer et al. 2001). In another study, restrictedto pediatric patients (n=54) at Johns Hopkins (Baltimore, Md., USA),cashew reactivity ranked fourth (11%) behind walnut (26%), almond (13%),and, pecan (13%), perhaps reflecting a delayed dietary introduction ofcashews relative to other nuts. (Ehrlich P. 1989)

A number of plant, animal and fungal allergenic proteins have beenidentified by biochemical and molecular genetic means in recent yearsreflecting the hope that the detailed information provided by suchstudies will lead to the development of hypoallergenic foods, newtherapeutic and diagnostic tools, and an increased understanding of theproperties that render some proteins more allergenic than others.(Heisset al. 1999; Valenta R et al. 1999; Bannon et al. 2001; Niederberger etal. 2001; Bhalla et al. 2001)

Our previous screening of a cashew nut cDNA library with humancashew-allergic sera primarily yielded clones encoding an IgE-reactivevicilin-like protein, designated Ana o1 (Wang et al. 2002). Here wedescribe the product of the single identified clone encoding alegumin-family (11S globulin) seed storage protein, designated Ana o 2.

The legumins are large (300-450 kD), generally hexameric,non-glycosylated members of a complex family of proteins. Individualsubunits are found both as intact precursor proteins (50-60 kD) and asprocessed acidic (30-40 kD) and basic (20 kD) subunits that generallyremain covalently associated in seeds. In addition to Ana o 2, severalother legumins have been identified as food allergens. These include Arah 3 (Rabjohn et al. 1999) and Ara h 4 (Kleber-Janke et al. 1999) of thepeanut, the soybean G1 and G2 glycinins (Zeece et al. 1999; Helm et al.2000a; Beardslee et al. 2000), Cor a 9 of the hazelnut (Beyer et al.2002), and possibly a legumin from buckwheat (Fujino et al. 2001; Yamadaet al. 1995). The legumins are related to each other structurally andare believed to share ancestors in common with vicilin-like 7S proteins.The vicilins, or 7S globulins, are also multimeric, digestion resistant,seed storage molecules found in many edible plants and are frequentlyallergenic.

More than 50% of the sera from cashew allergic patients (13 of 21, 62%)reacted with rAna o 2 formally making this a major allergen. Six of thesera reacted strongly (29%). Because of the known complexity of thelegumin family, and the differences observed between the singlerecombinant cashew legumin cDNA-derived sequence reported here and thesequenced cashew peptide fragments from IgE-reactive proteins previousreported (Teuber et al. 2002) it would not be unexpected if additionalreactivity to legumin epitopes not represented on our cloned andexpressed version were yet to be discovered. Nevertheless, at least forthe patient's serum tested, it appears that the recombinant proteinfully inhibited reactivity to the native extracted protein suggestingthat most or all members of the cashew legumin family share commonallergenic epitopes. However, further investigations comparingadditional cloned versions the legum ins to each other and to highlypurified native molecules for reactivity to human IgE will be needed toadequately determine if this Ana o 2 clone expresses the full range ofcashew legumin allergenic epitopes. The unique IgE-binding patterns ofeach pool of sera to the Ana o 2 linear epitopes suggests aheterogeneity of recognition patterns between patients though somepeptides were recognized by members of two or all three pools.

Linear epitope mapping has shown that the soybean G1 glycinin acidicchain may share cross-reactive epitopes with the peanut Ara h 3(Beardslee et al. 2000). Both epitopes mapped from the G1 acidic chainshow a considerable degree of identity (60% and 70%) with theoverlapping region of the homologous Ara h 3 epitopes though differenceswere noted in the critical amino acids.(Beardslee et al. 2000) It hasbeen shown directly that the IgE-binding regions of the soybean G2glycinin also bind IgE from peanut sensitive patients [Helm 2000]. Suchfindings are not surprising when one considers that both are members ofthe legume family and patients allergic to one have serum IgE antibodiesthat immunologically cross-react with other legumes. (Eigenmann et al.1996). While no such direct analysis has been performed to determine ifthere is cross-reactivity between our cashew legumin (Ana o 2) and thatof peanut (Ara h 3) and soybean (G1 and G2 glycinin), the BLASTalignment of these protein sequences and subsequent analysis of thepositional relationship and degree of homology between linear epitopeson each protein has been informative. The major findings are that thereis considerable positional overlap and some shared sequence identifybetween Ana o 2 and soybean G2 glycinin but little in common between Anao 2 and peanut Ara h 3 linear epitopes. We have shown that the majorityof Ana o 2 linear epitopes (68% overall and 83% of the most stronglyreactive) are on the acidic chain of the protein. Previous studies havealso observed stronger reactivity with the acidic chain rather than thebasic chain of allergenic legumins [Pederson 1989](Herian et al. 1990;Rabjohn et al. 1999; Beardslee et al. 2000)[Helm 2000].

It is worth noting that the majority of Ana o 2 epitopes are located inthe same areas of the protein sequence that is evolutionarily leastconserved with respect to insertions and deletions among a variety oflegumins (compare FIG. 2C with the acidic chain of Ana o 2 in FIG. 3);features that would further minimize cross-reactivity between plantlegumins. It should also be borne in mind though that these variousresults represent only those reactions between a subset of leguminsequences in each of the compared plant seeds and the IgE from a limitedset of patients. The inclusion of additional legumins and a broader poolof patient sera in future analyses could shed further light on thesevarious relationships.

Clinical information regarding cross-reactivity between cashews andlegumes has not been well documented, but based on our findings we canpredict that while there may be a basis for cross-reactivity betweencashew and soybean, it is unlikely that such cross-reactivity existsbetween cashew and peanut legumins, at least with regard to linearepitopes. Similarly, we previously reported that cross-reactivitybetween the vicilin linear epitopes of cashew (Ana o 1) and peanut (Arah 1) would also be unlikely (Wang et al. 2002), observations which,together, further explains the lack of cross-reactivity between treenut- and peanut-reactive patients' sera (Sicherer et al. 2001).

To date, no common structural characteristics of linear-IgE epitopeshave been identified (reviewed in (Bredehorst and David 2001)), but ourfinding that all four legumins analyzed share an epitope with positionaloverlap, suggests that epitope mapping can provide important clues tothe nature of allergenicity. Three-dimensional modeling of our cashewlegumin is continuing and when compared to the models of G2 glycinin(Helm et al. 2000b) and other homologous allergens, may provideadditional insights.

The Various Aspects of the Claimed Invention

The present invention thus discloses isolated nucleic acid sequences,polypeptide products thereof, and associated methods. The skilled willrecognize that the isolated nucleic acids will be useful at least whenexpressed in a suitable cell or organism to produce the encodedpolypeptides, which in turn may be employed in testing to identifypatients allergic to cashew nuts. Furthermore, expression of the nucleicacid sequences of the present invention in a suitable cell may be usefulin studying and characterizing gene function.

Accordingly, the present invention includes an isolated nucleic acidsequence comprising the nucleotide sequence of SEQ ID NO:1, or adegenerate variant of SEQ ID NO:1. SEQ ID NO:1 is the coding sequencefor the described Ana-o-2 cashew antigen, including a stop codon “TAA”at positions 1372-1374. The nucleic acid sequence described may alsocomprise at least one mutation selected from a deletion, a substitution,and an addition. A preferred embodiment of the invention includes anexpression vector comprising the nucleic acid sequence noted aboveoperably linked to an expression control sequence, and a cultured cellcomprising this vector, the cell, or a progeny of the cell, preferablybeing transfected with the vector, wherein the cell expresses apolypeptide encoded by said nucleic acid sequence. The nucleic acid ofSEQ ID NO:1 may also further comprise additional nucleotides accordingto SEQ ID NO:2, which provides a tail for the coding sequence.Additionally, the invention further includes an isolated nucleic acidcomprising a sequence that encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:3. An additional aspect of the inventionincludes a method of producing a protein by culturing the cellcontaining nucleic acid sequence SEQ ID NO: 1 under conditionspermitting expression of a polypeptide encoded by the nucleic acid. Themethod may also include purifying the polypeptide from the cell or themedium of the cell.

The invention also discloses twenty-two (22) linear polypeptideepitopes, as listed in Table III, which are also respectively listedherein as SEQ ID NO:4 through SEQ ID NO:25. Accordingly, the inventionincludes an isolated nucleic acid comprising a sequence that encodes apolypeptide comprising an amino acid sequence selected from SEQ IDNOS:4-25. Additionally claimed is a purified Ig-E binding polypeptidethe amino acid sequence of which comprises at least one sequenceselected from SEQ ID NOS:4-25, and wherein the polypeptide comprises atleast one mutation in said selected amino acid sequence, the mutationbeing selected from a deletion, a substitution, an addition, andparticularly a mutation which reduces the polypeptide's IgE-bindingcapacity but retains at least some immunogenicity. Those skilled in theart should recognize that the scope of the invention includes anisolated nucleic acid sequence or a degenerate variant thereof, whichencodes a polypeptide consisting essentially of an amino acid sequenceselected from SEQ ID NOS:4-25, and that the selected amino acid sequencemay include at least one mutation such as a deletion, a substitution, oran addition. It should be understood that by “essentially” it is meantthat the polypeptide is largely, but not wholly, the specified sequence.

Further method aspects of the disclosed invention include a test fordetecting a cashew allergy in a patient, said test comprising contactingthe patient with an amount of a purified polypeptide comprising at leastone amino acid sequence selected from SEQ ID NOS:3-15. In this test,contacting preferably comprises introducing the purified polypeptideinto the patient, for example, by injection. The skilled will know thatinjecting includes any of the known routes useful in immunologicaltesting of patients, for example, intradermally, and subcutaneously.Additionally, the purified polypeptide for use in such a test maycomprise at least one mutation in said amino acid sequence, and themutation may be selected from a deletion, a substitution, and anaddition.

Yet a further diagnostic test of the invention for detecting anti-cashewIgE in a patient to thereby indicate an allergy to cashews, the testcomprising several steps. The patient's serum is reacted with a purifiedpolypeptide the amino acid sequence of which comprises at least onesequence selected from SEQ ID NOS:4-25. The polypeptide is separatedfrom unreacted patient serum, and the polypeptide is reacted with alabeled human IgE-reactive agent after separating from unreacted patientserum. The polypeptide is then separated from unreacted labeled humanIgE-reactive agent, and the labeled human IgE-reactive agent bound tothe polypeptide after separating from unreacted agent is detecteddirectly or indirectly to thereby indicate presence in the patient'sserum of anti-cashew IgE. The amino acid sequence of the polypeptide foruse in this test may also comprise at least one mutation selected from adeletion, a substitution, and an addition.

The invention also includes a test for screening patients for allergy tocashews which may be embodied in a test kit comprising a first reagentcontaining at least one purified polypeptide whose amino acid sequencecomprises at least one sequence selected from SEQ ID NOS:4-25. The testkit preferably also includes a second reagent containing at least onelabeled human IgE-reactive agent. Additionally, in the test kit thefirst reagent may include a solid phase carrying the purifiedpolypeptide.

The skilled will find additional guidance in carrying out the inventionby consulting Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, N.Y., and also Ausubel et al. (eds.),1995, Current Protocols in Molecular Biology (John Wiley & Sons, N.Y.).In addition, guidance in diagnostic and screening test methods suitablefor use with the polypeptides of the present invention may be found inManual of Clinical Laboratory Immunology, Sixth Edition, Rose et al.(eds.), 2002, American Society for Microbiology, Washington, D.C. Asnoted above, these publications are incorporated herein in theirentirety.

Accordingly, in the drawings and specification, there have beendisclosed typical preferred embodiments of the invention, and althoughspecific terms are employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

Sequence Listings

Following is a written sequence listing for nucleic acid SEQ ID NOS: 1-2and for polypeptides SEQ ID NOS:3-25. Also enclosed is a completesequence listing in computer readable form. The information recorded onthe form is identical to the written sequence listing.

1-11. (canceled)
 12. A purified Ig-E binding immunogenic polypeptide theamino acid sequence of which comprises at least one sequence selectedfrom SEQ ID NOS:4-25.
 13. The polypeptide of claim 12, furthercomprising at least one mutation in said selected amino acid sequence.14. The polypeptide of claim 13, wherein said at least one mutation isselected from a deletion, a substitution, and an addition.
 15. Thepolypeptide of claim 12, further comprising at least one mutation insaid amino acid sequence which reduces the polypeptide's IgE-bindingcapacity.
 16. A test for detecting cashew allergy in a patient, saidtest comprising contacting the patient with an amount of a purifiedpolypeptide comprising SEQ ID NO:3.
 17. A test for detecting a cashewallergy in a patient, said test comprising contacting the patient withan amount of a purified polypeptide comprising at least one amino acidsequence selected from SEQ ID NOS:4-25.
 18. The test of claim 17,wherein contacting comprises introducing the purified polypeptide intothe patient.
 19. The test of claim 17, wherein the purified polypeptidecomprises at least one mutation in said amino acid sequence.
 20. Thetest of claim 19, wherein said at least one mutation is selected from adeletion, a substitution, and an addition.
 21. An in vitro diagnostictest for detecting anti-cashew IgE in a patient, said test comprising:reacting the patient's serum with a purified polypeptide the amino acidsequence of which comprises at least one sequence selected from SEQ IDNOS:3-25: separating the polypeptide from unreacted patient serum;reacting the polypeptide with a labeled human IgE-reactive agent afterseparating from unreacted patient serum; separating the polypeptide fromunreacted labeled human IgE-reactive agent; and detecting labeled humanIgE-reactive agent bound to the polypeptide after separating fromunreacted agent to thereby indicate presence in the patient's serum ofanti-cashew IgE.
 22. The diagnostic test of claim 20, wherein said aminoacid sequence further comprises at least one mutation selected from adeletion, a substitution, and an addition.
 23. A test kit for screeningpatients for allergy to cashews, said kit comprising a first reagentcontaining at least one purified polypeptide whose amino acid sequencecomprises at least one sequence selected from SEQ ID NOS:3-25.
 24. Thetest kit of claim 23, further comprising a second reagent containing atleast one labeled human IgE-reactive agent.
 25. The test kit of claim23, wherein said first reagent further comprises a solid phase carryingthe purified polypeptide. 26-27. (canceled)